Diabetes is a chronic disease that afflicts 200 millions people in the world. Type 1 diabetes results from autoimmune destruction of Beta cells, while type 2 diabetes is caused by a combination of insulin resistance and inadequate insulin secretion. Thus, in both type 1 and type 2 diabetes, the functional Beta cell mass is not sufficient to control glycemia. The mature pancreas contains two types of tissue: exocrine tissue composed of acinar cells that produce enzymes (e.g., carboxypeptidase-A) secreted via the pancreatic ducts into the intestine and endocrine islets composed of cells that produce hormones such as insulin (Beta cells), glucagon (alpha cells) somatostatin (delta cells) and pancreatic polypeptide (PP cells). Over the past decades research in the Beta cell field profited from the establishment of insulin-secreting cell lines, such as RIN and INS1 cells derived from x-ray induced rat insulinoma (Asfari et al., 1992; Gazdar et al., 1980), HIT cells generated by transformation of hamster islet cells by SV40 (Santerre et al., 1981) and BetaTC and Min6 cells derived from transgenic mice expressing SV40 T antigen under the control of the insulin promoter (Efrat et al., 1995; Efrat et al., 1993; Efrat et al., 1988; Hanahan, 1985; Knaack et al., 1994; Miyazaki et al., 1990). Such cell lines were useful for a better understanding of Beta cell biology and could be used for drug screening.
Generation of pancreatic Beta cells in large amount represents an important objective for at least 2 reasons: first such Beta cells would be useful for screening of new drugs that can modulate Beta cell function; next such pancreatic Beta cells could be used for cell therapy of diabetes. To this end, different approaches have been previously developed to generate pancreatic Beta cells in large amount.
The first one consisted in using as starting material immature stem cells (ES cells) to produce mouse or human Beta cells. The major advantage is that ES cells self-renew indefinitely in culture, and have the capacity to differentiate to multiple cell types, and thus to pancreatic Beta cells. While quite a large amount of publications appeared during the past years on Beta cells production from ES cells, (Assady et al., 2001; Blyszczuk et al., 2003; Brolen et al., 2005; Hori et al., 2002; Lumelsky et al., 2001; Soria et al., 2000), other publications described pitfalls in such works, questioned the interpretations and demonstrated that reproducible protocols were not yet available to produce Beta cells from ES cells (Hansson et al., 2004; Rajagopal et al., 2003).
Thus, at that point, functional Beta cells have not yet been generated in large quantities from ES cells with the exception of one recent publication where Beta cells developed from hES cells (D'Amour et al., 2006). However, such cells did not secrete insulin upon glucose stimulation.
The second approach was based on the production of pancreatic Beta cell lines using pancreas as a starting material. There, two main approaches have been followed. In the first case, adult Beta cells were transformed. This was performed either by x-ray induced rat insulinoma (Asfari et al., 1992; Gazdar et al., 1980), or by transformation of hamster islet cells by SV40 (Santerre et al., 1981) and more recently by immortalization of adult human Beta cells with SV40 LargeT antigen and human telomerase reverse transcriptase. While some cell lines were generated from adult Beta cells, the efficiency of the approach was extremely low. For example, while large efforts were developed to generate human Beta cell lines form adult islets (de la Tour et al., 2001; Demeterco et al., 2002; Gueli et al., 1987; Ju et al., 1998; Levine et al., 1995; Soldevila et al., 1991), only one human Beta cell line was developed (Narushima et al., 2005). The functional human Beta cell line NAKT-15 published in Narushima et al. represented a step toward a potential cure of diabetes by transplantation (Narushima et al., 2005). However, as indicated in this paper, among 253 clones analyzed, only one expressed insulin and transcription factors featuring Beta cells. This method is thus not amenable for obtaining large scale mature Beta cells for diagnosis or therapy.
Another approach was to derivate Beta cell lines from Beta cell tumours derived from transgenic mice expressing SV40 T antigen under the control of the insulin promoter (Efrat et al., 1995; Efrat et al., 1993; Efrat et al., 1988; Hanahan, 1985; Knaack et al., 1994; Miyazaki et al., 1990). However, because such Beta cell lines were obtained by gene transfer in fertilized eggs, its application is restricted to animal models without any possible transfer to human.
Recently, we demonstrated that immature pancreas infected with recombinant lentiviruses resulted in endocrine cell differentiation and restricted cell type expression of the transgene according to the specificity of the promoter used in the viral construct. Specifically, when eGFP was placed under the control of the insulin promoter, a majority of the developed Beta cells expressed eGFP. (Castaing et al., 2005b). Thus, recombinant lentiviral vectors can efficiently infect pancreatic progenitor cells and thereby stably modify mature rat pancreatic Beta cells. In addition, we asked whether Beta cell lines can be generated by infecting pancreatic progenitor cells that will differentiate into Beta cells. For this purpose, we infected immature rat or human pancreatic tissues with recombinant lentiviruses expressing SV40 largeT antigen and/or hTERT under the control of the insulin promoter. Our data demonstrate that recombinant lentiviruses can infect both rat and human pancreatic stem/progenitors, that will differentiate into Beta cells expressing the transgenes and form insulinoma from which Beta cell lines can be derived. For this purpose, rat immature pancreatic epithelia were transduced with recombinant lentiviruses expressing the SV40 LargeT antigen under the control of the insulin promoter. Such infected tissues were next transplanted under the kidney capsule of immuno-incompetent mice. Such environment had previously been shown to be permissive for the development of many organs such as ovarian cortex, thyroid, skin and airway (Delplanque et al., 2000; Levy et al., 1998; Martin et al., 1993; Weissman et al., 1999). We also demonstrated that pancreatic Beta cells also properly developed from rat or human immature pancreases under such conditions (Castaing et al., 2005a; Castaing et al., 2005b; Castaing et al., 2001).
In connection with the present invention, our objective was to define new approaches to generate functional Beta cell lines in sufficient quantity to provide cell therapy treatment.
We continued the investigations to maximize amplification of master cell batches of mature rat pancreatic Beta cells and we tried to apply the above method to generate master cell batches of human pancreatic Beta cells. Unfortunately, as of today, we never observed any formation of insulinoma with human cells contrary to what was observed when rat immature pancreases are infected with the same virus months after transplantation. Moreover, when we dissociated and cultured the infected cells, we were unable to generate human cell lines.
We also directly infected the cells with viruses expressing the hTert under the control of the insulin promoter together with recombinant lentiviruses expressing the SV40 LargeT antigen under the control of the insulin promoter and again, using conditions identical to the ones used to generate rat Beta cell lines, we were unable to generate human Beta cell lines.
We thus had to define a new strategy for gene transfer into Beta cell tumors to generate human Beta cell lines. In course of this work, we discovered that using a sub-graft protocol, we were able to form insulinoma-structure with human functional Beta cells and that the sub-grafting steps led to the specific enrichment in Beta cells ultimately leading to a homogenous human Beta cell lines which can be further amplified to clinical and commercial scale.
Accordingly, we now have at hand a method for specifically establishing and amplifying human Beta cells and not other cell types. By repeating enrichment and amplification steps, we were able obtain repeatedly cell lines which can be amplified for testing, diagnosis or therapeutic use.
Using the above sub-transplantation procedure to enrich the graft in proliferating beta cells, we were able to generate 11 independent human beta cell lines. Such lines express insulin and have a gene expression profile that resembles to adult beta cells. In addition, when transplanted under the kidney capsule of diabetic mice they were able to normalize blood glucose. the human beta cell lines are able to normalize glycemia of diabetic mice. By performing intraperitoneal glucose load these animals were able to utilize normally the glucose load, demonstrating their insulin secretion capabilities. Moreover, by performing glucose tolerance test in vivo on transplanted diabetic mice, we have been able to demonstrate that our cell line is able to respond to glucose stimulation and therefore is fully functional.
Finally, our human beta cell lines can be efficiently used to detect the presence of auto-antibodies found in sera of diabetic patients and thereby have a great potential for diagnosis of type I diabetes.
These Beta cells are now being used to generate and amplify ad infinitum human Beta cell lines which form master cell batches for diagnostic. This also opens perspective towards clinical use of Beta cells in the treatment of diabetes.